CZ390597A3 - Peptides and process for preparing thereof - Google Patents

Abstract

The invention pertains to medicine, specifically to methods of obtaining biologically active substances with immunoregulatory properties, and can be used in medicine, veterinary science and experimental biochemistry. The problem addressed by the invention is that of producing a novel synthetic biologically active peptide with immunoregulatory properties of formula X-A-D-Trp-Y, in which A is D-Glu, iD-Glu; X is H or Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D-Tyr, D-Phe, D-Trp, gamma -aminobutyric acid, xi -aminocaproic acid; Y is Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D-Tyr, D-Phe, D-Trp, gamma -aminobutyric acid, xi -aminocaproic acid, -OH or substituted amide (C1-C3). The method of producing the peptide involves the synthesis of glutamil-containing peptides in solution by scission of an internal anhydride of tributyloxycarbonyl glutamic (D or L) acid with the appropriate k-salt of D-Trp-Y. This is followed by chromatographic separation of alpha - and gamma -isomers. Further synthesis was carried out by building up a peptide chain using activated esters of tributyloxycarbonyl amino acids.

Description

Peptides and their preparation

Technical field

The invention relates to medicine.

methods for preparing biologically active substances having inunoregulatory properties and can be used in medicine, veterinary medicine and experimental biology.

BACKGROUND OF THE INVENTION

The importance of developing novel innocuous and munoregulatory peptides that would stop the progressive growth of such diseases.

such as malignant neoplasms, sepsis, chronic and latent infections, developing against the background of immunodeficiency states, have been demonstrated by a large number of studies in this field.

The most common method of developing new peptides is to isolate active peptide fractions from tissue extracts, fractionators and purification, including isolating and identifying units (SU 1600047, SU 1638849, SU 1737798).

Thymus extracts as regulators of immune processes, particularly thymosin fraction 5 (Goldstein A.L., Guna A.,

Latz M.M., Hardy H.A.,

White A.) and thyamalin (CH 6595 86).

The extracts contain substances of a polypeptide nature whose production from natural raw materials is limited by the complexity of the process, the low yields of the active substances and the great variety of their physical and chemical characteristics as well as the biological properties.

In addition, ballast elements present in natural thymus (thymus) preparations sometimes cause side effects.

The latter circumstance becomes synthetic peptides. So far, a large number of immunoregulatory peptides have been synthesized by the stimulus to create:

PCT / WO 089/06134 by SU

1541821,

5013723.

SU 1518956, EP 230052, EP 406931, US 5021551

Each of the synthetic peptides has a limited combination of essential properties, is highly active, low toxic, does not cause any side effects and can therefore be used in medicine.

The object of the present invention was to synthesize novel biologically active peptides having an immunoregulatory effect.

Another object of the invention was to develop a completely new peptide synthesis technology which had a minimum of simple steps and resulted in high yields of the products of the following structure, which is critical in the pharmaceutical industry.

SUMMARY OF THE INVENTION

The present invention provides polypeptides of the general formula

X-A-D-Trp-Y wherein

A is D-Glu or iD-Glu,

X is H or Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D- Tyr, D-Phe, D-Trp, β-aminobutyric acid and -amino-caproic acid,

Y is Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D-Tyr, D-Phe, D-Trp, γ-aminobutyric acid, N-aminocaproic acid, -OH or substituted C 1 -C 3 amide.

The present invention also provides a process for the preparation of the peptides as defined above, which comprises synthesizing glutamyl-containing peptides in solution by opening the internal anhydride of tributyloxycarbonylglutamine (D or L) acid using the appropriate D-Trp-Y derivative followed by a- and τ- isomers. Further synthesis is accomplished by peptide chain building using activated tri-butoxycarbonylamino esters.

Table 1 gives Rfi (in chloroform-methanol-32% acetic acid 60 = 45 = 20) and Rf2 (in butanol-pyridine-water-acetic acid 5: 5 = 4 = 1) for some new analogues peptides.

-3Table 1

Peptide Rfl R * Abu-D-Glu-D-Trp-OH 0.32 0.53 Abu-1D-Glu-1-Tip-OH 0.29 0,49 | Aca-D-Glu-D-Trp-OH 0.31 0.52 Acai D-Glu-D-Trp-OH 0.28 0.49 Ala-D-Glu-D-Trp-OH 0.34 0.61 Ala-1D-Glu-D-Trp-OH 0.32 0.55 D-Ala-D-Glu-D-Tip-D-Ala 0.22 0.41 D-Ala-D-Glu-D-Trp-OH 0.26 0.51 D-Ala-1D-Glu-D-Txp-D-Ala 0.25 0.50 D-Ala-iDGlu-DTrp-OH 0.25 0.51 D-Ile-D-Glu-D-Tip-D-Leu 0.35 0.53 D-Ile-Ddu-D-Trp-OH 0.36 0.54 D-He-ID-Glu-D-Trp-D-Leu 0.34 0.53 D-non-iD-Glu-D-Tro-OH 0.27 0.S7-- .. .... D - Leu - D - Glu - D - Tip - OH 0.36 0.53 DLeuD-Glu-DTrpOH 0.35 0.52 D-NVal-D-Glu-D-Trp-OH 0.33 0.51 D-NVal-iD-Glu-D-Trp-OH 0.32 0.51 DPhe-D-Glu-DTrp-Ala 0.37 0.56 D-Phe-1D-Glu-D-Trp-Ala 0.36 0.55 DProD-Glu-D-TrpOH 0.38 0.58 D-Pro-1D-Glu-D-Trp-OII 0.37 0.58 D-Trp-D-Glu-D-TrpOH 0.41 0.59 D-Trp-1D-Glu-D-Trp-OH 0.40 0.59 D-Tyr-D-Glu-D-Trp-D-Tyr 0.39 0.58 D-Tyr-1D-Glu-D-Trp-D-Tyr 0.38 0.58 D-Val-D-Glu-D-Trp-OH 0.34 0.51 D-Val-iD-Glu-D-Trp-OH 0.33 0.50 Gly-D-Glu-D-Trp-NVal 0.29 0.51 Gly-D-Glu-D-Trp-OH 0.30 0.54 Gly-iD-Glu-D-Trp-NVal 0.32 0,53 | Gly-1D-Glu-D-Trp-OH 0.29 0,53 |

····

Peptide Rfl Rfz H-D-Giu-D-Tip ~ Abu 0.33 032 H-D-Glu-Trp-Aca 0.30 0.53 H-D-Glu-D-Trp-D-Ile 0.35 0.58 H - D - Glu - D - Tip - D - Pro 0.37 »« H-D-Glu-D-Trv-Glv  . . C, '29 J 0/54 | u-D-Trp-He 0.36 0.58 H-D-Glu-D-Trp-Leu 0.36 039 H-D-GJuDTrp-Ly 0.28 0,47 | H-D-Glu-DTrp-OH 0.36 0.56 H-D-Gh-D-Trp-Phe 0.39 0.61 HD-Giu-DTrp-Pro 0.38 039 H-D-Glu-D-Trp-Tyr 0.37 038 H-D-Glu-D-Trp-Val 0.38 0.61 H-iDGlu-DTrp-Abu 0.34 031 H-iDGlu-DTrp-Aca 0.33 030 H-D-Glu-D-Trp-D-Ile 0.34 035 H-iD-Glu-D-Trp-D-Pro 0.36 038 H-iD-GluD-Trp-Gly 0.28 0.52 H-iD-GluD-Trp-Ue 0.34 0.57 H-iD-Glu-D-Trp-Leu 0.35 038 H-iD-Glu-D-Trp-Lys 0.27 0.45 H-D-Glu-D-Trp-OH 0.30 032 HiDGlu-D-Txp-Fhe 0.38 0.61 H-ID-Glu-D-Trp-Pro 0.37 0.60 1 H-iD-Glu-D-Trp-Tyr 0.35 0.58 H-iD-Glu-DTrp'V _and l 0.36 0.60 Ile-D-Glu-DTrp-D-Phe 0.42 0.68 Ile-D-Glu D-Trp-OH 0.38 0.56 Ile-iGlu-D-Trp-D-Phe 0.40 0.67 Ile-iD-GluDTrp-OH 0.37 035 Leu-D-Glu-DTrp-OH 0.35 0.62 LeuiDGlu-DTrp-OH 0.27 0.57 NVal-D-Glu-D-Trp-OH 0.35 0.61 NVal-iD-Glu-D-Trp-OH 0.34 0.60

Fepticl · Rfl Phe-D-Glu-D-Trp-OH 0.41 0.63 Phc-iD-Glu-D-ΌΗρΌΗ 0.40 0.62 Pro-D-Glu-D-Trp-OH 0.43 0.63 Pro-ID-Glu-D-Trp-OH 0.42 0.62 Trp-D-Glu-DTrp-OH 0.45 0.68 Trp-ID-Glu-D-Trp-OH 0.44 0.67 Tyr-D-Glu-D-Trp-OH 0.42 0.64 Tyr-1D-Glu-D-Trp-OH 0.41 0.63 Val - D - Glu - D - Trp - D - Val 0.46 0.68 Val-D-Glu-D-Trp-OH 0.33 0.50 Val-iD-Glu-D-Trp-D-Val 0.44 0.66 Val-iD-Glu-D-TrpOH 0.34 0.52

• • •

The following example illustrates the invention in more detail.

DETAILED DESCRIPTION OF THE INVENTION

H-D-Glu-D-Trp-OH; and HiD-Glu-D-Trp-OH

1. Preparation of Boc-D-Glu-OH

Dissolve 14.7 g (0,1 M) of H-D-Glu-OH in 200 ml of distilled water. The pH was adjusted to 10.2 using 1M KOH and a solution of 33.0 g (0.3 M) of BOC2O in dioxane was added with vigorous stirring. The pH is controlled using the pH-state. After completion of the reaction, the mixture is transferred to a separatory funnel, extracted from alkaline with 3x 150 ml of ethyl acetate, slowly added to the aqueous phase with a 0.2% sulfuric acid solution until a pH of 3 is reached, and then Boc-D-Glu-OH is extracted. into an aqueous phase (3 x 200 mL). The organic layer was washed with saturated aqueous solution (3 x 200 mL) to neutral pH, dried over Na 2 SO 4 and then evaporated under reduced pressure to an oil. The yield is

16.7 g (68%).

2. Preparation of a mixture of Boc-D-Glu-Trp-OH and Boc-iD-Glu-D-Trp-OH

Boc-D-Glu-OH (16.7 g, 0.068 M) was dissolved in dimethylformamide (200 ml), cooled to 0 ° C and a solution of N, N -dicyclohexylcarbodiimide (20.6 g, 0.1 Μ) in dimethylformamide (100 ml) was added with stirring. The mixture was stirred at +4 ° C for 4 hours and then allowed to stand at room temperature for 8 hours. The precipitated dicyclohexylcarbamide was collected by filtration and washed with 2 x 50 ml dimethylformamide. The filtrate was concentrated by evaporation under reduced pressure to 1/2 volume, and 24.3 g (0.1 M) of H-D-Trp-OH was added thereto while cooling to +4 ° C with vigorous stirring. The solution was then allowed to warm to room temperature. Completion of the reaction was checked by thin layer chromatography (TLC) in a chloroform · ethyl acetate methanol 6 = 3: 1 disappearance of the spot.

internal Boc-D-Glu-anhydride. The rest of dihexylcarb- am i du se The reaction mixture is separated by filtration, the dimethyl formamide is evaporated off reduced 200 ml

ethyl acetate and 200 ml of 0.2% sulfuric acid solution. The organic layer was separated, washed to neutral pH with a saturated solution, and washed with a saturated solution.

Na 2 SO 4 and dried over Na 2 SO 4 -E ethyl acetate solution was added

evaporate under reduced pressure. The resulting oily precipitate is a mixture of Boc-D-Glu-D-Trp-OH and Boc-1D-Glu-D-Trp-OH. Total yield 25.4 g (70%).

3. Preparation of H-D-Glu-D-Trp-OH and H-iD-Glu-D-Trp-OH

Dissolve 25.4 g of the mixture (0.048 M) in 200 ml of form

and stirred for 1 h at 40 ° C. The solvent was evaporated in vacuo

under reduced pressure to give a thick oil containing peptides, which were separated and purified by ion exchange chromatography in a column packed with Sephadex in a gradient of 0.01-0.2M pyridine acetate buffer (buffer). The yield was 5.7 g (35%) of H-D-Glu-D-Trp-OH and 5.7 g (35%) of H-D-Glu-D-Trp-OH.

The following physical and chemical characteristics of the peptide are determined.

Primary structure H-D-Glu-D-Trp-OH and H-D-Glu-D-Trp-OH Empirical formula C16H20N3O5 Molecular weight 334.35 Appearance yellow-white or gray powder Solubility freely soluble in water, slightly in alcohol, insoluble in chloroform UF spectrum in the range of 250 to 300 nm it shows a minimum at 280 ± 2 nm and an arm at 287 ± 2 nm. Biological activity The novel peptide is tested in mice Balb / c. Spleen cells mice are suspended in RPMI 1640 with 2 ml glutamine and 5% inactivated fatal serum and

they are then plated in shallow dishes at 100 µl (200,000 cells) per dish. The preparation to be examined is introduced at the beginning of the cultivation. Concanavalin A is used as a mitogen at a final concentration of 2 meg per plate. Plates are incubated at 37 ° C in 5% CO 2 for 48 hours. Cell proliferation is assessed after 3H-thymidine, which is added at a dose of 5 mcg / ml 24 hours before the end of the culture, by scintillation counting and expressed in terms of disintegration per minute (CPM). The results are shown in Table 2.

Table 2

Preparation C P Μ I Prepata dose, mcg / ml 1 5 10 20 May iD-Glu-D-Trp 68594 63428 20043 13222 Cyclosporin A 67649 2698 574 569 Control 61467

^x> mean of three measurements

The novel peptides were found to inhibit the proliferation of spleen cells.

To determine the safety of the peptides, an acute toxicity study was conducted according to the methodological recommendations of the Pharmacological Committee of the Russian Federation for the requirements of preclinical studies of the overall toxic effect of new pharmacological agents.

Based on the results obtained, it was found that at a 100-fold intraperitoneal dose of the peptides, there was no acute toxic effect and the LD50 could not be achieved with these doses.

Utility of the invention

Biologically active peptides can be widely used in medicine and veterinary medicine.

Claims (2)

PATENT CLAIMS 1. Peptides of the general formula X-A-D-Trp-Y wherein A is D-Glu or iD-Glu, X is H or Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D- Tyr, D-Phe, D-Trp, γ-aminonic acid and -anocaproic acid, Y is Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D-Tyr, D-Phe, D-Trp, ^am i NOMAS 1 to acids, aminocaproic acid, -OH or C1-C3 substituted amide. 2. A process for preparing peptides of the general formula X-A-D-Trp-Y wherein A is D-Glu or iD-Glu, X is H or Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D- Tyr, D-Phe, D-Trp, aminobutyric acid and -amino-caproic acid, Y is Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D-Tyr, D-Phe, D-Trp, -g ¹ -amino-nomasic acid, -amino-caproic acid, -OH or substituted C 1 -C 3 amide, characterized in that it comprises the steps of peptide synthesis in solution to open tributyloxycarbonylglutaminic anhydride (D or L) ) acid using D-Trp-Y K-salt, further chain building using activated esters and mixed anhydrides, with prior cleavage of the tributyloxycarbonyl group by treatment with formic acid followed by chromatographic separation of α- and β-isoners.